Recent discoveries have raised expectations that stem cells may be a source of replacement cells and tissues that are damaged in the course of disease, infection, or because of congenital abnormalities. Various types of putative stem cells differentiate when they divide, maturing into cells that can carry out the unique functions of particular tissues, such as the heart, the liver, or the brain. A particularly important discovery has been the development of pluripotent stem cells, which are thought to have the potential to differentiate into almost any cell type.
Early work on pluripotent stem cells was done in mice (reviewed in Robertson, Meth. Cell Biol. 75:173, 1997; and Pedersen, Reprod. Fertil. Dev. 6:543, 1994). Mouse stem cells can be isolated from both early embryonic cells and germinal tissue. Desirable characteristics of pluripotent stem cells are that they be capable of indefinite proliferation in vitro in an undifferentiated state, retain a normal karyotype, and retain the potential to differentiate to derivatives of all three embryonic germ layers (endoderm, mesoderm, and ectoderm).
The development of preparations of human pluripotent stem cells has involved overcoming a number of technical difficulties, and is considerably less advanced than work with mouse cells.
Thomson et al. (U.S. Pat. No. 5,843,780; Proc. Natl. Acad. Sci. USA 92:7844, 1995) were the first to successfully isolate and propagate pluripotent stem cells from primates. They subsequently derived human embryonic stem (hES) cell lines from human blastocysts (Science 282:114, 1998). Gearhart and coworkers derived human embryonic germ (hEG) cell lines from fetal gonadal tissue (Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998; and U.S. Pat. No. 6,090,622).
Both hES and hEG cells are reported to have the long-sought characteristics of pluripotent stem cells: they are capable of long-term proliferation in vitro without differentiating, they have a normal karyotype, and they remain capable of producing a number of different cell types. Because of this, they hold considerable promise for use in human therapy, acting as a reservoir for regeneration of almost any tissue compromised by genetic abnormality, trauma, or a disease condition.
A significant challenge to the use of pluripotent stem cells for therapy is that they are traditionally cultured on a layer of feeder cells to prevent differentiation (U.S. Pat. No. 5,843,780; U.S. Pat. No. 6,090,622). Without feeder cells in the culture environment, hPS cells soon die, or differentiate into a heterogeneous population of committed cells. Leukemia inhibitory factor (LIF) inhibits differentiation of mouse PS cells, but it does not replace the role of feeder cells in preventing differentiation of human PS cells. Unfortunately, using feeder cells increases production costs, impairs scale-up, and produces mixed cell populations that require the pluripotent stem cells to be separated from feeder cell components.
Another challenge is to control differentiation of stem cells into the particular type of tissue required for treatment of each patient. It is a hypothesis of this invention that better understanding of the differentiation process will be obtained by observing gene expression during growth and differentiation of pluripotent stem cells.
International Patent Publication WO 99/20741 (Geron Corp.) is entitled Methods and Materials for the Growth of Primate-Derived Primordial Stem Cells. A cell culture medium is provided for growing primate-derived primordial stem cells in a substantially undifferentiated state, having a low osmotic pressure and low endotoxin levels. The basic medium is combined with a serum effective to support the growth of primate-derived primordial stem cells on a substrate of feeder cells or a feeder cell matrix. The medium further includes non-essential amino acids, an anti-oxidant, and growth factors that are either nucleosides or a pyruvate salt.
Sequence-based studies of early human development have focused on libraries produced from fetal organs and tissues for example, fetal libraries from the I.M.A.G.E. consortium; http://image.llnl.gov/). International Patent Publication WO 98/00540 (Incyte) reports partial sequences of stem cell antigens, isolated from cDNA libraries derived from THP-1 cells and bladder tumors.
New technology to facilitate growing and manipulating undifferentiated pluripotent stem cells would be a major achievement towards realizing the full potential of embryonic cell therapy.